Microfluidic surface-mediated emulsion stability control

ABSTRACT

A microfluidic emulsion droplet generation system and methods of use thereof are provided. The system may include a microfluidic substrate having a flow path configured and arranged for emulsion droplet generation, at least one textured surface in the flow path configured and arranged for inducing surface-mediated coalescence of emulsion droplets; and at least one channel junction in the flow path for emulsion droplet formation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 62/378,490, filed Aug. 23, 2016, which isincorporated herein by reference in its entirety.

BACKGROUND

The field of microfluidics has advanced to the point that it isfulfilling much of its promise to supplant conventional laboratory fluidhandling. The ability to precisely control the movement, accession,allocation, and mixing of minute amounts of fluids and subject thosefluids to additional processing, analysis, and the like has helped movethe field into the mainstream of scientific research, diagnostics, andmedical devices.

As research and diagnostic needs become more and more complex, however,there is a need for the field of microfluidics to similarly advance incomplexity, requiring a wide range of new functionalities within themicrofluidic context. By way of example, microfluidic systems have beenused to deliver and combine reagents within microfluidic channels andthen perform subsequent processing and/or analytical operations on thosereagents, including, e.g., thermal cycling, separations, optical,chemical or electrical detection, and a host of other operations.

In other applications, microfluidic systems have been used to partitionsmall aliquots of aqueous fluids within flowing streams of immisciblefluids, e.g., oils, in order to compartmentalize reactions within thosepartitions for separate processing, analysis, etc. Specificimplementations of these systems have been used to compartmentalizeindividual nucleic acids in order to perform quantitative amplificationand detection reactions (qPCR).

In another implementation, the GEMCODE™ system from 10× GENOMICS®,discrete droplets in an emulsion contain both template nucleic acids andbeads bearing large numbers of oligonucleotide barcodes, where a givenbead will have a constant barcode sequence. The barcode is then used toprime replication of fragments of the template molecules within theparticular partition. The replicate fragments created within a givendroplet will all share the same barcode sequence, allowing replicatefragments from single long template molecules to be attributed to thatlonger template. Sequencing of the replicate fragments then providesbarcode linked-reads that can be later attributed back to an originatinglong fragment, provide long range sequence context for shorter sequencereads.

Surface wettability of substrates is an important physical property forthe design of microfluidic droplet-based assays. Surface wettability caninfluence droplet generation as well as droplet/emulsion stability.Typically, the surface wettability and emulsion stability is tuned byusing surfactants in the dispersed and continuous phases. Surfacewettability can also be controlled by coatings and chip materials used.Additionally, the surface roughness/texture also influences emulsionstability especially when the droplets interact with surfaces such as incollection wells of the chips. In particular, roughness induced wettingof surfaces can cause large scale coalescence of emulsion and therebyfailed assays.

With increasing demands on microfluidic systems, there is a need to addto the microfluidic tools that can be applied to expand their utility.The present disclosure provides a number of such tools and the uses andapplications thereof.

SUMMARY

In general, in one embodiment a microfluidic emulsion droplet generationsystem is provided including: a microfluidic substrate having a flowpath configured and arranged for emulsion droplet generation; at leastone textured surface in the flow path configured and arranged forinducing surface-mediated coalescence of emulsion droplets; and at leastone channel junction in the flow path for emulsion droplet formation.

In one aspect the at least one textured surface is disposed in the flowpath downstream of the channel junction.

In another aspect the substrate further comprises one or moremicrochannels or reservoirs in the flow path upon which the at least onetextured surface is disposed. In a particular aspect the reservoir is anoutlet well.

In a further aspect the at least one textured surface in the flow pathof the system is a microtexture or a nanotexture surface. In aparticular aspect the at least one textured surface is produced in thesubstrate by injection molding, photolithography, embossing or anycombinations thereof. In yet another aspect the at least one texturedsurface is textured by nano-pillars, nano-cones, nanofibers, nanotubes,microgrooves, striations, tool marks, coatings or any combinationsthereof. In a further aspect the at least one textured surface isconfigured in an array. In another aspect the at least one texturedsurface provides for spontaneous wetting, superhydrophobicity,superoleophobicity, interfacial slip or any combinations thereof.

In one embodiment one or more cross-sectional dimensions of the flowpath are less than 200 microns. In another aspect one or morecross-sectional dimensions of the flow path are less than 100 microns.In yet another aspect one or more cross-sectional dimensions of the flowpath are less than 50 microns.

In general, in another embodiment, a method of emulsion dropletformation using the droplet generation system and aspects thereof asdescribed above is provided including: providing a dispersed phase and acontinuous phase to the system; and forming emulsion droplets comprisingthe dispersed phase and continuous phase in the system.

In one aspect of the method droplet formation is performed without asurfactant in the dispersed phase or continuous phase. In a specificaspect the dispersed phase is aqueous and the continuous phase comprisesoil.

In another aspect of the method the at least one textured surface isdisposed in the flow path downstream of the channel junction.

In a further aspect of the method the substrate further includes one ormore microchannels or reservoirs in the flow path upon which the atleast one textured surface is disposed. In a particular aspect thereservoir is an outlet well.

In another aspect of the method the at least one textured surface is amicrotexture or a nanotexture. In one aspect the at least one texturedsurface is produced in the substrate by injection molding,photolithography, embossing or any combinations thereof. In anotheraspect the at least one textured surface is textured by nano-pillars,nano-cones, nanofibers, nanotubes, microgrooves, striations, tool marks,coatings or any combinations thereof. In a further aspect the at leastone textured surface is configured in an array. In yet a further aspectthe at least one textured surface provides for spontaneous wetting,superhydrophobicity, superoleophobicity, interfacial slip or anycombinations thereof.

In a further aspect of the method one or more cross-sectional dimensionsof the flow path are less than 200 microns. In one aspect one or morecross-sectional dimensions of the flow path are less than 100 microns.In another aspect one or more cross-sectional dimensions of the flowpath are less than 50 microns.

In another aspect of the method the emulsion droplets includepolynucleotides, barcodes, beads or combinations thereof. In oneembodiment the polynucleotides and barcodes are attached to the beads.In another embodiment the bead includes a covalent bond that iscleavable upon application of a stimulus. In a particular embodiment thecovalent bond is a disulfide bond.

In general, in another aspect a method of emulsion dropletsurface-mediated coalescence using the system described above includes:providing a dispersed phase and a continuous phase to the system foremulsion droplet formation; forming emulsion droplets in the system;directing the emulsion droplets to the textured surface; and coalescingthe emulsion droplets.

In one aspect the surface-mediated coalescence is achieved without achemical agent coalescence stimulus. In another aspect the emulsiondroplets are coalesced after a reaction performed in the emulsiondroplets.

In one embodiment of the method the reaction is a polymerase chainreaction (PCR).

In another embodiment of the method the at least one textured surface isdisposed in the flow path downstream of the channel junction.

In yet another embodiment of the method the substrate further includesone or more microchannels or reservoirs in the flow path upon which theat least one textured surface is disposed. In one aspect the reservoiris an outlet well.

In another embodiment of the method the at least one textured surface isa microtexture or a nanotexture. In one aspect the at least one texturedsurface is produced in the substrate by injection molding,photolithography, embossing or any combinations thereof. In anotheraspect the at least one textured surface is textured by nano-pillars,nano-cones, nanofibers, nanotubes, microgrooves, striations, tool marks,coatings or any combinations thereof. In a further aspect the at leastone textured surface is configured in an array. In another aspect the atleast one textured surface provides for spontaneous wetting,superhydrophobicity, superoleophobicity, interfacial slip or anycombinations thereof.

In another embodiment of the method one or more cross-sectionaldimensions of the flow path are less than 200 microns. In a particularaspect one or more cross-sectional dimensions of the flow path are lessthan 100 microns. In a further aspect one or more cross-sectionaldimensions of the flow path are less than 50 microns.

In another embodiment of the method the emulsion droplets comprisepolynucleotides, barcodes, beads or combinations thereof. In one aspectthe polynucleotides and barcodes are attached to the beads. In anotheraspect the bead comprises a covalent bond that is cleavable uponapplication of a stimulus. In a further aspect the covalent bond is adisulfide bond.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretiesfor all purposes and to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photomicrographs of microfluidic outlet wellsincluding rough and smooth surface textures respectively and containingemulsion droplets according to example embodiments of the presentinvention.

FIGS. 2A and 2B are illustrations of a microfluidic feature includingrough and smooth surface textures in series, and showing their effect onemulsion droplets according to an embodiment of the present invention.

FIGS. 3A-3C are topography images showing example surfaces having smoothsurfaces according to embodiments of the present invention.

FIGS. 4A-4C are topography images showing example surfaces havingroughened surfaces according to embodiments of the present invention.

DETAILED DESCRIPTION I. General Overview Emulsion Droplets and ReagentPartitioning

The present disclosure provides devices, systems, and methods that are,in some embodiments, particularly useful in managing complex samples foranalysis using high throughput analytical systems, including, forexample, high throughput nucleic acid analysis systems, such as nucleicacid arrays, nucleic acid sequencing systems, nucleic acid amplificationand quantitation systems, or the like. In particular, certainembodiments of the devices, systems, and methods described herein areparticularly useful in providing encapsulated reagents or reagentsystems, and co-partitioning these reagents with sample components forfurther reaction and/or analysis. This co-partitioning of reagents andsample components can be used, for example, in reducing the complexityof the sample material by segregating portions of the sample todifferent partitions. Further, by also segregating reagents, one cansubject each sample portion to a different reaction, including forexample, the application of unique identifiers to different samplecomponents, e.g., attachment of a discrete barcode or tagging reagentsto the discrete sample components.

Particularly elegant examples of these co-partitioning approaches aredescribed in Published international Patent Application No.WO2014/028537, and U.S. Patent Application Publication Nos. US20140206554, US 20140227684, US 20140228255, and US 20150292988 the fulldisclosures of each of which are incorporated herein by reference intheir entirety for all purposes.

By way of example, one particularly elegant approach provides a polymermicrocapsule composition that includes nucleic acid barcode sequencesbound to the microcapsule, where the barcodes associated with a givenmicrocapsule have substantially the same sequence of nucleotides, butwhere different discrete microcapsules will have different barcodesequences associated with such microcapsules. Each of thesemicrocapsules is then contacted with a portion of a sample fluid, suchas a sample fluid that includes a template nucleic acid from a samplematerial. The mixture of sample material including the template nucleicacid and the microcapsule is then partitioned into a small volume, suchas a droplet in a water in oil emulsion, such that the microcapsule anda portion of the sample material are contained within the same droplet.In addition to controlling the droplet generation process to provide adesired number of microcapsules in a given partition, the samplematerial and emulsion process also may be controlled to provide for adesired amount of sample material, e.g., sample nucleic acid material,within each partition, e.g., to provide a single template molecule or adesired level of genome coverage within a given partition, or otherdesired level of sample materials.

Within the partition, the barcode sequence is reacted with the samplematerial, e.g., the template nucleic acid to effectively tag the samplematerial or a portion thereof. For example, by reacting the barcodesequence with the template, e.g., through amplification of the templatesequence using the barcode sequence as an extension primer, one caneffectively “attach” the barcode sequence to the replicated or amplifiedtemplate. Similarly, replication of the extended primer produces acomplement of the template along with a complement to the barcode,again, effectively attaching the barcode to the template. The presenceor attachment of the barcode sequence, or its complement, on or to theamplified template molecule, or its complement, then allows some levelof attribution of sequence reads that include that barcode to the sameportion of sample material, e.g., the same template molecule or the samesample components, that was originally allocated to that partition.

In many cases, the molecule that includes the barcode sequence orsequences may also include functional elements that are used insubsequent processing of the amplified template sequences. Thesefunctional sequences include, for example, primer sequences e.g.,targeted or universal), primer recognition sequences, sequences that canform secondary structures, either within the sequence, or uponreplication of the sequence, enrichment sequences, e.g., that are usedas affinity purification sequences, immobilization sequences, probesequences, reverse complement or hairpin sequences, or any of a varietyof other functional sequences.

There are a wide variety of other high-value applications for suchpartitioning and barcoding or tagging processes. The present disclosureadvantageously provides devices, systems and methods that can greatlyfacilitate the generation of such partitioned compositions or componentsthereof.

II. Fluidic Systems for Producing Encapsulated Reagents and PartitionedReactions

The present disclosure provides improved fluidic systems, andparticularly improved microfluidic systems, that are useful for both thegeneration of encapsulated reagents, as well as in the partitioning ofthose encapsulated reagents for use in subsequent reactions and/oranalyses. As used herein, microfluidic systems typically denote fluidicsystems that employ one or more fluid conduits, channels, chambers, orthe like that include one or more interior cross-sectional dimensions,e.g., depth, length or width, that are less than 1000 microns, less than200 microns, less than 100 microns, and in some cases, less than about50 microns, or even less than about 20 microns. In some cases, one ormore cross-sectional dimensions may be about 20 microns or less or 10microns or less. Typically, these microfluidic channels or chambers willhave at least one cross-sectional dimension of between about 1 and about100 microns.

As will be appreciated, reference to encapsulated reagents is notintended to limit the scope of such reagents to completely enclosedcapsules, but is intended to reflect any of a variety of methods ofassociating reagents with a given particle, bead, or other solid orsemi-solid particle phase. In particular, encapsulation generally refersto the entrainment or other attachment, coupling, or association of aparticular species with a solid or semi-solid particle, bead, enclosure,partition or droplet, and is not limited to compositions in which thespecies is entirely or partially enclosed within a larger structure.

In some aspects, encapsulated reagents are associated with microcapsulesthat are generally spherical in shape, although they may be elongated,plug shaped, or otherwise vary in their specific shape. In some cases,microcapsules will have one or more cross-sectional dimensions that areless than 200 microns, less than 150 microns, or less than about 100microns. In some cases, microcapsules of the present disclosure have oneor more cross-sectional dimensions that are between about 10 and about200 microns, between about 20 and 150 microns, between about 30 and 125microns, in many cases between about 40 and about 100 microns, and stillother cases, between about 50 and about 75 microns.

While the dimensions of the microcapsules can be an importantconsideration, in many applications the variability in those dimensionsis also an important consideration. In particular, for example, thetransport of a microcapsule through a microfluidic system can besignificantly impacted by the size of that microcapsule. For example,simple flow resistance may be greater for much larger microcapsules thanfor smaller microcapsules. Similarly, propensity for clogging may begreater for larger microcapsules than for smaller microcapsules. Ineither event, flow rates of microcapsules through a microfluidic systemmay be greatly impacted by the size of the microcapsule. Accordingly, incertain aspects, the microcapsules of described herein, will be providedas a population of microcapsules having substantially monodispersecross-sectional dimensions. In terms of cross-sectional dimensions, thephrase substantially monodisperse refers to a population that deviatesexpressed as a coefficient of variation and stated as a percentage) fromthe mean cross-sectional dimension by no more than 50%, no more than40%, no more than 30%, no more than 20%, or in some cases, no more than10%.

Whether in the context of generating microcapsules for use inentrainment or encapsulation of reagents, or in the partitioning ofaqueous fluids within non-aqueous droplets, the devices and systems ofthe present disclosure can employ a similar architecture in someembodiments. In a simplified example, this architecture in someembodiments may include a first channel segment that is fluidlyconnected to a first junction that fluidly connects the first channelsegment with a second channel segment and a third channel segment. Thesecond channel segment delivers to the junction a second fluid that isimmiscible with the first aqueous fluid, such as an oil, that allows forthe formation of aqueous droplets within the stream of immiscible fluid.This second fluid may be referred to herein as the dispersion fluid,partitioning fluid or the like. The flow of the first and second fluidsthrough the junction and into the third channel segment is controlledsuch that droplets of the first fluid are dispensed into a flowingstream of the second fluid within the third channel segment.

A variety of modifications to this basic structure according toembodiments of the present invention are available to better controldroplet formation and to bring in additional fluid streams. As usedherein, the control of fluid flows encompasses both active control offluid flows through the application of greater or lesser driving forcesto cause that fluid flow. Additionally, flows may be controlled in wholeor in part, by controlling the flow characteristics of one or more ofthe fluids and/or the conduits through which they are flowing. Forexample, in some embodiments, fluid flow may be controlled by providinghigher flow resistance within a conduit, e.g., through providing ahigher viscosity, narrower conduit dimension, or providing larger orsmaller microcapsules within a fluid stream, or any combination of theforegoing. In some cases, control is imparted through several ofcontrolled driving force, controlled conduit dimensions, and controlledfluid properties, e.g., viscosity or particle composition.

In microfluidic droplet generation, surfactants are frequently used toprevent the coalescence of droplets to maintain partitioning ofreagents. However, in some cases, it is desirable to cause coalescenceto occur, such as when bulk biochemical reactions or cleanups of thechemical species of droplets are to be performed after in-dropletreactions. There are various means to cause coalescence, including butnot limited to—electric fields, and destabilizing surfactants. However,each of these methods require the addition of chemicals or additionalcomplexity in the form of active elements (such as electrodes forelectro-coalescence) in the system. It is sometimes desirable to causecoalescence by passive means. Macro-fluidic example: liquid-liquidseparations (Agarwal, S. et al. Separation and Purification Technology107:19-25 (2013)).

Types of Roughness—Amplitude/Frequency

In general, increasing the roughness of a surface causes a hydrophilicsurface to become more hydrophilic and a hydrophobic surface to becomemore hydrophobic (Quere, D. Annu. Rev. Mater. Res. 38:71-99 (2008); seealso Zhao, M. et al. Sci. Rep. 4, 5376; DOI:10.1038/srep05376 (2014)).The roughness of a surface can be described by the amplitude and thefrequency or density of the features causing an increase in the surfacearea. The frequency of the roughness can be particularly influential onthe wettability of a surface, with a higher frequency causing a surfaceto be more wettable than a lower frequency. According to embodiments ofthe present invention, this increased wettability can be used topurposefully cause coalescence of microfluidically-generated droplets onthe surface. In some embodiments, the roughness may have a frequency ofup to one surface feature (e.g., peak) per nanometer distance along thesurface. In some embodiments, the roughness may have a frequency of lessthan one surface feature per nanometer distance along the surface (e.g.,one surface feature per 10 nm, 100 nm, 1 ∥m, etc.). The frequency of theroughness, according to some embodiments, may range from 0.001 to 1features per nm along the surface. The surface features may have anamplitude (e.g., height) in the nano- or micrometer scale. In someembodiments, the amplitude of the surface features is less than 100 μm.In some embodiments, the amplitude of the surface features is less than50 μm. In some embodiments, the amplitude of the surface features isless than 10 μm. In sonic embodiments, the amplitude of the surfacefeatures is less than 1 μm. In some embodiments, the amplitude of thesurface features at least 10 nm. In some embodiments, the amplitude ofthe surface features may range from about 10 nm to about 10 μm, fromabout 10 nm to about 1 μm, from about 10 nm to about 500 nm, or fromabout 10 nm to about 100 nm. In some embodiments, the amplitude of thesurface features may range from about 50 nm to about 5000 nm, from about50 nm to about 1000 nm, from about 50 nm to about 500 nm, from about 50nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nmto about 100 nm. For example, in FIGS. 1A and 1B, the amplitude of theroughness of both exit wells are on the order of 100 nm. However, thefrequency of roughness in FIG. 1A, in which coalescence on the wellsurface occurs, is higher than that of FIG. 1B.

Methods of introducing textured surfaces in a microfluidic substrateaccording to some embodiments can include, but are not limited toinjection molding, photolithography, embossing, sanding, machining, andsurface coatings or any combinations thereof. In some embodiments, thesurface is textured to have a regular pattern of surface features (e.g.,an evenly spaced array of surface features). In other embodiments, thesurface may be randomly patterned with surface features such that thedistance between individual surface features may vary. In someembodiments, the surface features on a roughened surface may bedifferently sized/shaped or, in other embodiments, may be similarlysized/shaped. Methods of removing surface roughness include polishing,etching, and surface coatings or any combinations thereof.

Surface texturing can be in the form of, for example, providing definednano-pillar/cones/nanofibers/nanotubes arrays, microgrooves, striations,tool marks or coatings. In some embodiments, it is also possible andadvantageous to provide directional wetting implemented by surfacetexturing. In some embodiments, direction wetting may be achieved byproviding anisotropic nano-scale (e.g., 10 nm to 1000 nm) features on asurface. In some embodiments, the anisotropic features are arrangedassymetically on the surface to provide directional wetting. In someembodiments, a the surface may be patterned with the features describedby Kuang-Han Chu et al., “Uni-directional liquid spreading on asymmetricnanostructured surfaces,” Nature Materials 9, 413-417 (2010), which isincorporated by reference herein in its entirety. In some embodiments,directional wetting can be used as a passive valving mechanism whichallows wetting of substrates in a desired direction and not in others.This can allow robust priming and reagent loading to the chip byensuring a well defined initial condition of reagent locations in thechannels.

III. EXAMPLES Example 1: Surface-Mediated Coalescence of EmulsionDroplets in Microfluidic Features

Surface-mediated coalescence of emulsion droplets containing gel beads(GEMs) was tested in a microfluidic chip substrate including outletwells for GEMs collection. The GEMs were made up of a gel bead in anaqueous phase surrounded by oil. The aqueious phase included a buffer,enzymes, glycerol, cell lysis surfactant; the oil phase included oil andemulsion-stabilizing surfactant. The microfluidic chips were molded froma thermoplastic material. In a first case, outlet wells weremanufactured with a rougher surface texture than in a second case wherethe outlet wells had a smoother surface texture. Surface roughness ofthe well was determined by the roughness of the mold used to shape thethermoplastic from Which the chips were made, which is in turn wasdetermined by the machining process used to make the mold. Polishing themold (resulting in smoother surfaces) prevented coalescence. Accordingto white-light interferometry (WLI) measurements, there was no regularpattern to the roughness, the Rt=maximum peak−minimum valley was 0.7μm-3 μm. Example topography images of the surfaces are shown in FIGS.3A-4C. FIGS. 3A-3C show topography images of relatively smoother examplesurfaces which were non-coalescing. FIGS. 4A-4C show topography imagesof example surfaces with rougher surface texture Which resulted incoalescence.

The same number of GEMs were introduced into each type of outlet well.The effect of surface texture on GEMs stability was observed byphotomicroscopic viewing of the bottoms of the outlet wells. The effecton stability was visualized by observing the amount of intact vs.coalesced GEMs through the bottom of the outlet wells.

FIGS. 1A and 1B are photomicrographs of the experimental results. FIG.1A shows an outlet well having a rougher surface texture and GEMscollected therein. FIG. 1B shows a different outlet well having asmoother surface texture and GEMs collected therein. By comparing thetwo images, it is readily apparent that the GEMs in FIG. 1A are fewer innumber as a result of surface-mediated coalescence of the GEMs. Pooledaqueous phase evidencing coalescence of the GEMs in FIG. 1A is much moreapparent at the outer edges of the outlet wells than in FIG. 1B.

The results of this study indicated that rougher surface texturecorrelates with increased coalescence of GEMs, while smoother surfacetexture supports stability of GEMs. It can be concluded from theseresults that emulsion droplets, for example GEMs, can be effectivelymanipulated by microfluidic feature surface roughness/smoothness design.Both stabilization of the emulsion droplets as well as surface-mediatedcoalescence can be achieved as desired based on application of differentsurface textures to the microfluidic feature surfaces and interactionwith emulsion droplets.

Example 2: Controlled Emulsion Droplet Surface-Mediated Coalescence

In a microfluidic emulsion droplet generation system or device, surfacetexturing of microfluidic channels, wells or other physical features canprovide improved control over emulsion droplet formation. Depending onthe desired application, microchannels, wells or other features in asubstrate can be configured for superhydrophobicity (providinganti-wetting characteristics) or superhydrophilicity (providing wettingcharacteristics), which could benefit droplet generation, preservationand coalescence. This could be achieved, for example, by use ofnano/micro-structured surface texture within the entire or a portion ofthe microchannels.

One potential application of this approach is to generate and preservestable emulsion droplets even in the presence of detergents which wouldotherwise wet the native microchannel substrate and favor coalescence.Application of surface texture design including smooth to varyingdegrees of roughness could be used to control emulsion droplet stabilityeven in the presence of detergents or other agents.

An exemplary design for controlling emulsion droplet surface-mediatedcoalescence in a microfluidic substrate according to embodiments of thepresent invention is illustrated in FIGS. 2A and 2B. Microfluidicfeature 200 can be, for example, a fluid path, channel, well etc. of amicrofluidic system. The microfluidic feature 200 in the exampleillustrated includes two distinct regions 201, 202 of surface texture.It can be understood that any of a number of configurations would bepossible, including two or more distinct regions of surface texture, andspacing of textured surfaces as required for controlling stability ofemulsions droplets or for coalescence. Region 202 is relatively smoothin texture. Region 201 is relatively rough in texture in comparison toregion 202. The microfluidic feature 200 further includes oil 204 thatcan be flowed in the feature 200 to transport emulsion droplets 203 inthe feature 200.

As shown in FIG. 2A at a first time point the amount of oil 204 providedkeeps the emulsion droplets 203 positioned in the smooth surface textureregion 202. In this state, the emulsion droplets 203 are stable and canbe controlled in such a state while remaining in proximity of the region202. In FIG. 2B, additional oil 204 has been introduced and the emulsiondroplets 203 were driven into the rough surface texture region 201 ofthe microfluidic feature 200. As illustrated in FIG. 2B,surface-mediated coalescence results in loss of the intact emulsiondroplets 203 and formation of coalesced emulsion droplets 205.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications may be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A microfluidic emulsion droplet generation systemcomprising: a) a microfluidic substrate having a flow path configuredand arranged for emulsion droplet generation; b) at least one texturedsurface in the flow path configured and arranged for inducingsurface-mediated coalescence of emulsion droplets; and c) at least onechannel junction in the flow path for emulsion droplet formation.
 2. Thesystem of claim 1, wherein the at least one textured surface is disposedin the flow path downstream of the channel junction.
 3. The system ofclaim 1, wherein the substrate further comprises one or moremicrochannels or reservoirs in the flow path upon which the at least onetextured surface is disposed.
 4. The system of claim 3, wherein thereservoir is an outlet well.
 5. The system of claim 1, wherein the atleast one textured surface is a microtexture or a nanotexture.
 6. Thesystem of claim 1, wherein the at least one textured surface is producedin the substrate by injection molding, photolithography, embossing orany combinations thereof.
 7. The system of claim 1, wherein the at leastone textured surface is textured by nano-pillars, nano-cones,nanofibers, nanotubes, microgrooves, striations, tool marks, coatings orany combinations thereof.
 8. The system of claim 1, wherein the at leastone textured surface is configured in an array.
 9. The system of claim1, wherein the at least one textured surface provides for spontaneouswetting, superhydrophobicity, superoleophobicity, interfacial slip orany combinations thereof.
 10. The system of claim 1, wherein one or morecross-sectional dimensions of the flow path are less than 200 microns.11. The system of claim 1, wherein one or more cross-sectionaldimensions of the flow path are less than 100 microns.
 12. The system ofclaim 1, wherein one or more cross-sectional dimensions of the flow pathare less than 50 microns.
 13. A method of emulsion droplet formationusing the droplet generation system of claim 1 comprising: a) providinga dispersed phase and a continuous phase to the system; and b) formingemulsion droplets comprising the dispersed phase and continuous phase inthe system.
 14. The method of claim 13, wherein droplet formation isperformed without a surfactant in the dispersed phase or continuousphase.
 15. The method of claim 13, wherein the dispersed phase isaqueous and the continuous phase comprises oil.
 16. The method of claim13, wherein the at least one textured surface is disposed in the flowpath downstream of the channel junction.
 17. The method of claim 13,wherein the substrate further comprises one or more microchannels orreservoirs in the flow path upon which the at least one textured surfaceis disposed.
 18. The method of claim 17, wherein the reservoir is anoutlet well.
 19. The method of claim 13, wherein the at least onetextured surface is a microtexture or a nanotexture.
 20. The method ofclaim 13, wherein the at least one textured surface is produced in thesubstrate by injection molding, photolithography, embossing or anycombinations thereof.
 21. The method of claim 13, wherein the at leastone textured surface is textured by nano-pillars, nano-cones,nanofibers, nanotubes, microgrooves, striations, tool marks, coatings orany combinations thereof.
 22. The method of claim 13, wherein the atleast one textured surface is configured in an array.
 23. The method ofclaim 13, wherein the at least one textured surface provides forspontaneous wetting, superhydrophobicity, superoleophobicity,interfacial slip or any combinations thereof.
 24. The method of claim13, wherein one or more cross-sectional dimensions of the flow path areless than 200 microns.
 25. The method of claim 13, wherein one or morecross-sectional dimensions of the flow path are less than 100 microns.26. The method of claim 13, wherein one or more cross-sectionaldimensions of the flow path are less than 50 microns.
 27. The method ofclaim 13, wherein the emulsion droplets comprise polynucleotides,barcodes, beads or combinations thereof.
 28. The method of claim 27,wherein the polynucleotides and barcodes are attached to the beads. 29.The method of claim 27, wherein the bead comprises a covalent bond thatis cleavable upon application of a stimulus.
 30. The method of claim 29,wherein the covalent bond is a disulfide bond.
 31. A method of emulsiondroplet surface-mediated coalescence using the system of claim 1comprising: a) providing a dispersed phase and a continuous phase to thesystem for emulsion droplet formation; b) forming emulsion droplets inthe system; c) directing the emulsion droplets to the textured surface;and d) coalescing the emulsion droplets.
 32. The method of claim 31,wherein surface-mediated coalescence is achieved without a chemicalagent coalescence stimulus.
 33. The method of claim 31, wherein theemulsion droplets are coalesced after a reaction performed in theemulsion droplets.
 34. The method of claim 31, wherein the reaction is apolymerase chain reaction (PCR).
 35. The method of claim 31, wherein theat least one textured surface is disposed in the flow path downstream ofthe channel junction.
 36. The method of claim 31, wherein the substratefurther comprises one or more microchannels or reservoirs in the flowpath upon which the at least one textured surface is disposed.
 37. Themethod of claim 36, wherein the reservoir is an outlet well.
 38. Themethod of claim 31, wherein the at least one textured surface is amicrotexture or a nanotexture.
 39. The method of claim 31, wherein theat least one textured surface is produced in the substrate by injectionmolding, photolithography, embossing or any combinations thereof. 40.The method of claim 31, wherein the at least one textured surface istextured by nano-pillars, nano-cones, nanofibers, nanotubes,microgrooves, striations, tool marks, coatings or any combinationsthereof.
 41. The method of claim 31, wherein the at least one texturedsurface is configured in an array.
 42. The method of claim 31, whereinthe at least one textured surface provides for spontaneous wetting,superhydrophobicity, superoleophobicity, interfacial slip or anycombinations thereof.
 43. The method of claim 31, wherein one or morecross-sectional dimensions of the flow path are less than 200 microns.44. The method of claim 31, wherein one or more cross-sectionaldimensions of the flow path are less than 100 microns.
 45. The method ofclaim 31, wherein one or more cross-sectional dimensions of the flowpath are less than 50 microns.
 46. The method of claim 31, wherein theemulsion droplets comprise polynucleotides, barcodes, beads orcombinations thereof.
 47. The method of claim 46, wherein thepolynucleotides and barcodes are attached to the beads.
 48. The methodof claim 46, wherein the bead comprises a covalent bond that iscleavable upon application of a stimulus.
 49. The method of claim 48,wherein the covalent bond is a disulfide bond.